Mycobacterium bovis causes infection (tuberculosis) in a variety of farm and wild animals and also humans, Van Emden, et al., Iowa State University Press, Ames, Iowa (1995). This organism along with M. tuberculosis, M. africanum, and M. Microti belongs to the taxonomically closely related members of M. tuberculosis complex. Numerous attempts have been made to seek a species-specific antigen with high sensitivity for diagnosis of M. bovis infection. A protein antigen MPB70 is secreted from M. bovis cells following cleavage of a 30-amino acid signal peptide which directs active transport of the protein across the cytoplasmic membrane Terasaka, et al., FEMS Microbiol. Lett., 58:273-276 (1989). The protein forms a major component of M. bovis culture filtrate, accounting for up to 10% of the protein excreted by some M. bovis Bacillus Calmette-Guerin (BCG) strains, Harboe, et al., Infect. Immun., 52:293-302 (1986), and possibly as much as 23%, Abou-Zeid, et al., J. Gen Microbiol., 132:3047-3053 (1986). The function of this protein is unknown.
The protein was first purified to homogeneity from culture filtrates of M. bovis BCG by Nagai, et al., Infect. Immun., 31:1152-1160 (1981), and later from culture filtrates of M. bovis AN-5 by Fifis, et al. Also, the gene coding M. bovis MPB70 has been cloned, sequenced and expressed in Escherichia coli, Hewinson, et al., Scand. J. Immun., 26:445-454 (1987). The molecular mass of MPB70 was estimated to be 18-23 kDa by SDS-PAGE, 15 kDa by sedimentation equilibrium analysis, and 16 kDa as deduced from the gene-derived amino acid sequence. The MPB70 protein exists as a glycosylated or non-glycosylated form.
The MPB70 protein is considered to be a highly species-specific immunodominant antigen that contains at least three separate M. bovis-specific epitopes, although some cross-reactivity with Nocardia asteroides has been reported. The protein is an active component of purified protein derivative (PPD) tuberculin, Harboe, et al., J. Clin. Microbiol., 28:913-921 (1990), and is able to elicit a delayed-type hypersensitivity response and to stimulate T- and B-lymphocyte responses in M. bovis-infected animals. Because of high species-specificity and its immunodominant properties that stimulate antibody production in infected animals, MPB70 has been incorporated into ELISA for detection of anti-MPB70 antibodies which serve as an indicator of M. bovis infection.